Expression of mycobacterium tuberculosis induced SOCS3 and STAT3 and the implications on innate immunity in TB patients vs healthy contacts in high TB/HIV endemic setting: A cross-sectional analytical study

Background Mycobacterium tuberculosis (TB) remains a disease of global health concern and a leading cause of mortality arising from an infectious agent. Protective immunity to TB remains unclear. Suppressor of cytokine signaling-3 (SOCS3) and signal transduction and activator of transcription-3 (STAT3) genes have shown potential to influence innate immunity. We, therefore, explored the expression of SOCS3 and STAT3 and their implications on the innate immunity in TB patients and their healthy close contacts. Methods We recruited 72 TB patients and 62 healthy contacts from a high TB and HIV endemic setting (Lusaka, Zambia). We used RT-PCRT and flow cytometry to quantify the expression of SOCS, STAT3 and cytokines respectively. Data was analysed Stata version 14.0 and figures were developed in GraphPad prism version 9.1.0 (221). Assessment for associations for categorical and continuous variables was analysed using the Chi-square test and Mann-Whitney test respectively. Spearman’s rank correlation was used to evaluate the relationship between SOCS3 and IL-6. A p-value < 0.05 was considered statistically significant. Results Healthy contacts markedly expressed SOCS3 in both unstimulated and stimulated whole blood in comparison to TB patients (p <0.0001). STAT3 was elevated in TB patients in TB patients in stimulated blood only. IL-6 (P = < 0.0001) and IL-10 (P = <0.0001), were significantly expressed in Healthy contacts in comparison to TB patients. TNF-α (p = 0.044) were markedly elevated in TB patients in comparison to healthy contacts. IL-6 and SOCS3 correlated significantly in healthy contacts only (r = 0.429, p = 0.02). Conclusions Both SOCS3 and STAT3 are genes of importance in mounting protective innate immunity against TB. We propose that SOCS3 stimulation and inhibition of STAT3 as possible approaches in gene therapy and vaccine development for TB.


I Introduction of technology
qEASY qPCR Primer Pairs are designed using SBI's proprietary primer design algorithm. They are used for SYBR Green dye-based real-time PCR and designed according to the conserved region of all variants of a specific gene. At least one primer crosses the junction of adjacent exons to avoid amplification of genomic DNA directly and effectively. Our primer pairs cover all genes from human, mouse, rat and can be widely applied to the quantitative analysis of gene expression. cDNA used as templates, a single, correct-size band is produced in SYBR Green dye-based PCR with each pair of primers. Therefore, our primers have the characteristics with high specificity, high amplification efficiency, wide linear range and uniform reaction conditions. Each package for a specific gene is supplied with a lyophilized mixture of forward and reverse primers that can be used directly in SYBR Green dye-based real-time PCR after they are dissolved into ultrapure water.

Shipping and Storage information
Shipping and Storage conditions: Each primer package contains a lyophilized mix of forward and reverse primers for a specific target gene in a centrifugal tube and is shipped at room temperature.
Upon receipt, store them at -20℃ and avoid repeated freeze-thaw cycles. When stored under these conditions and handled correctly, the product can be kept stably for 12 months in lyophilized powder and 6 months in solution.
Information of target genes and primers refer to page 1.

III Use process
The mix of forward and reverse primers of a specific gene is lyophilized powder, which is attached to the wall of centrifugal tube. Before using, centrifuge the tube for a few seconds, then re-suspend the primer mix in 200 µl dH 2 O to make a final concentration of 10 μM. We recommend preserving them with small packages and storing them at -20℃. Avoid repeated freeze-thaw cycles.
The customer only needs to take into account the volume of PCR reaction system when using the primer pairs. Pipet a certain amount of dissolved primer mix to make a final concentration of 0.2 μM and use it with SYBR Green qPCR Mix of other companies. The methods in detail refer to the part IV "Experimental Procedures".
The product is suitable for detection by SYBR Green and not necessarily suitable for Taqman.

Amount
Reaction 4. Set up and run the program to carry out the PCR reaction according to the operating manual of machine(e.g. Light Cycler 480 Ⅱ). Generally three-step way is applied(annealing temperature is 60 ℃).
Step 1: pre-denaturation 95℃ 5min Step 3. Set up and run the program to carry out the PCR reaction according to the operating manual of machine (e.g. Light Cycler 480 Ⅱ). Generally three-step way is applied (annealing temperature is 60 ℃).
Step 1: Reverse transcription 42℃ 10min Step 1: Pre-degeneration 95℃ 30s Step (1)The screening process of primers: design three pairs of primers in conserved regions for each specific gene. Trying them with the SYBR Green mix reagent by the qPCR experiment. The protocols in detail refer to the two-step RT-PCR protocol in the part Ⅳ Experimental Procedures.
(2) Generation of the verification report: through analyzing the amplification curves and dissolution curves of three pairs of primers, selected the pair of primers with high specificity and sensitivity.

Validation Report
The amplification curve and dissolution curve of a specific gene in the qPCR experiment (cDNA and ddH 2 O as templates). The validation report refer to page 1.

Catalog Number: HP100128
Human STAT3 qPCR primer pair VI Notes